Oral compositions and route of administration for the delivery of a thylakoid extract

ABSTRACT

The present invention provides a new use for a thylakoid extract, that is for oral route of administration, and a composition comprising the thylakoid extract in adjunction with an acceptable carrier for oral administration. Besides the pharmaceutical use, the thylakoid extract enters the composition of food or food supplements, for its inocuity and its capacity to provide a diet enriched in anti-oxidants and anti-inflammatory compounds. Therefore, in accordance with the present invention is provided the use of a thylakoid extract in the making of an oral composition for treating or preventing a disease or disorder involving the formation of reactive oxygen species or inflammation. Also is provided a method for treating or preventing a disease or disorder involving the formal of reactive oxygen species or inflammation in an individual, which comprises the step of orally administering an effective dose of a thylakoid extract. Further is provided an oral composition comprising a thylakoid extract and a vehicle for oral ingestion or oral administration. An oral composition comprising purified thylakoids and a carrier for oral ingestion or oral administration, with the proviso that the carrier does not essentially consists of water, physiological saline or propylene glycol is also provided as food or a food supplement, or as a pellet, or encapsulated granules or powder. The carrier may be present in an amount of 0.01% to 95% (w/w) of the total composition. The purified thylakoids are present in an amount which achieves a dosage of 0.1 to 10 mg per Kg of a subject&#39;s body weight.

FIELD OF THE INVENTION

This invention relates to oral administration of a thylakoid extract orof compositions comprising same.

BACKGROUND OF THE INVENTION

Thylakoids are specialized membranes that are responsible forphotosynthesis in eukaryotes (plant & algae) and prokaryotes cells(bacteria). These photosynthetic organisms convert CO₂ to organicmaterial by reducing this gas to carbohydrates in a complex set ofreactions. Electrons for this reduction reaction ultimately come fromwater, which is then converted to oxygen and protons. Energy for thisprocess is provided by light, which is absorbed by pigments (primarilychlorophylls and carotenoids).

The initial electron transfer (charge separation) reaction in thephotosynthetic reaction center sets into motion a long series of redox(reduction-oxidation) reactions, passing the electron along a chain ofcofactors and filling up the “electron hole” on the chlorophyll, muchlike in a bucket brigade. All photosynthetic organisms that produceoxygen have two types of reaction centers, named photosystem I &photosystem II (PSI and PSII) both of which are pigment/proteincomplexes that are located in thylakoids membrane.

Recently a dynamic and intact thylakoid membrane extract having bothanti-oxidative and anti-inflammatory properties and its use incombination with other anti-inflammatory compounds have been describedin International patent publication numbers WO 01/49305 and WO 03/04042,respectively. The anti-oxidative and anti-inflammatory properties of thethylakoid extract have been demonstrated in in vitro, ex vivo, in situand in vivo studies. Specifically, the thylakoid extract has been shownto capture the noxious reactive oxygen species including singlet oxygenspecies and to modulate pro- and anti-inflammatory cytokines towardattenuation of inflammation.

In vivo, topical applications (direct application at site of injury) ofthe thylakoid extract have been shown to prevent or reduce theUV-induced skin damages in hairless mice and to decrease TPA-induced earinflammation in rats and mice as well as preventing damage to intestinalmucosa induced by TNBS or DSS in rats. Also, intraperitoneal injectionof the thylakoid extract has been shown to reduce carrageenan-inducedpaw oedema. However, today, no data has confirmed the potential use ofthe thylakoid extract as an oral anti-oxidative and/or anti-inflammatoryagent.

The present invention relates to the use of a thylakoid extract as anoral therapeutic agent.

SUMMARY OF THE INVENTION

The present invention provides a new use for a thylakoid extract, thatis for oral route of administration, and a composition comprising thethylakoid extract in adjunction with an acceptable carrier for oraladministration. Besides the pharmaceutical use, the thylakoid extractenters the composition of food or food supplements, for its innocuityand its capacity to provide a diet enriched in anti-oxidants andanti-inflammatory compounds.

Therefore, in accordance with the present invention is provided the useof a thylakoid extract in the making of an oral composition for treatingor preventing a disease or disorder involving the formation of reactiveoxygen species or inflammation. Also is provided a method for treatingor preventing a disease or disorder involving the formation of reactiveoxygen species or inflammation in an individual, which comprises thestep of orally administering an effective dose of a thylakoid extract.Further is provided an oral composition comprising a thylakoid extractand a vehicle for oral ingestion or oral administration.

Therefore, in accordance with the present invention is provided a use ofpurified thylakoids in the making of an oral composition for treating orpreventing a disease or disorder involving the formation of reactiveoxygen species or inflammation.

Further is provided the use of purified thylakoids in the making of anoral composition for preventing oxidative damages to components of thecomposition. In a specific embodiement, the oral composition is food ora food supplement. In another embodiment, the oral composition is amedication against oxidative damages, disorders or diseases.

Also is provided a method for treating or preventing a disease ordisorder involving the formation of reactive oxygen species orinflammation, in a subject, which comprises the step of orallyadministering an effective dose of purified thylakoids.

An oral composition comprising purified thylakoids and a carrier fororal ingestion or oral administration, with the proviso that the carrierdoes not essentially consists of water, physiological saline orpropylene glycol is also provided as food or a food supplement, or amedication in the form of a pellet, encapsulated granules orencapsulated powder.

The carrier may be present in an amount of 0.01% to 95% (w/w) of thetotal composition.

The purified thylakoids are present in an amount which achieves a dosageof 0.1 to 10 mg per Kg of a subject's body weight.

DESCRIPTION OF THE INVENTION

Demonstration will be made hereinbelow that the thylakoid extract(hereinbelow also referred to as “purified thylakoids” or “PCT”) isactive when orally administered. The extract can be formulated as aliquid composition (a non-lyophilized extract), a lyophilized extractreconstituted in water, physiological saline or any other solutioncompatible with oral administration, in propylene glycol (100% or lowerconcentrations) or as a solid composition (as is or in adjunction withpharmaceutically acceptable carrier for oral administration). Thylakoidscompositions essentially consisting of lyophilized thylakoids,thylakoids reconstituted in water or in saline as well as thylakoidspurified and obtained in propylene glycol have been disclosed in WO01/49305, although their use for oral administration was not disclosedin this reference.

The contents of all cited documents are incorporated by reference.

Excipients and carriers are widely used in the pharmaceutical field andare known to those skilled in the art. Amongst them, binding agents,disintegrating agents and/or fillers are of current use. The form takenby the product may also vary widely. Dry products comprise pellets, andpowders and granules in a free form or in capsules. Liquid products maycomprise lipids (oils and fats), stabilizers, emulsifiers, surfactants,polymers, and/or any colorant or flavoring additive to improve thetaste, the scent or the appearance of the composition.

Examples of binding agents include gelatine, cellulose, celluloseethers, amyloses, dextrose, polyglycols, tragacanth, pectins, alginatesand polyvinyl pyrrolidone (PVP).

Examples of desintegrating agents include starches, modified starches(sodium starch glycolate, starch 1500, . . .) pectins, betonite,cellulose, cellulose derivatives like carboxymethylcellulose (CMC),alginates, PVPs, ultraamylopectin, crosslinked PVP or crosslinked CMC(such as Ac-Di-Sol/FMC).

Examples of fillers include lactose, glucose, fructose, calciumphosphates, sulfates or carbonates, starch, modified starch, sugaralcohols such as sorbitol and mannitol, cellulose derivatives,saccharose, and/or microcrystaline cellulose.

Several types and selections of auxiliary substances forming carriersfor oral use are described for example in Journal of Pharmaceutical Sc.(1963), vol 52, from p.918 and following.

Preparation of spheroids comprising plant material is described in U.S.Pat. No. 5,733,551.

In general, the amount of active ingredient, that is the thylakoids, canextend from 1 ug to 1 g per day in one or more doses. In humans, a rangeof doses of 0.1 to 10 mg per Kg of body weight appears to be suitable.Therefore, for an averaged 70 Kg subject, a 5-10 mg to 500-1000 mg dailydosage regimen would be adequate. Examples of 200 mg pellets comprising20, 40 and 60% (40, 80 and 120 mg) thylakoids have been made and aredescribed hereinbelow. Pellets of 200 to 300 mg can be also made of purecompressed thylakoids (without any auxiliary agents).

This invention will be described herein below referring to specificexamples, embodiements and figures, the purpose of which is toillustrate the invention rather than to limit its scope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of enteral administration of thylakoids onTPA-induced ear oedema.

FIG. 2 shows the effect of enteral and oral administration of thylakoidson carrageenan-induced paw oedema.

FIG. 3 represents the dosage of pigments to evaluate the pigmentintegrity following the compression of thylakoids at differentpressures.

FIG. 4 shows the photosynthetic activity of the thylakoids followingcompression at different pressures.

FIG. 5 shows the pigment integrity of the thylakoids followingcompression in the presence of diverse polymers.

FIG. 6 shows the effect of various concentrations of thylakoids indiverse polymers on the thylakoids synthetic activity.

EXAMPLE 1 The Thylakoids are Active as Enteral and Oral Compounds

Methodology

Animals

Male Wistar rats (180-200 g) were used in the experiments. The animalswere purchased from Charles River Canada (St-Constant, Qc, Canada). Theanimals were housed in an environmentally (t=25° C.) and air humidity(60%) controlled room with a 12 h light-dark cycle, kept on a standardlaboratory diet and drinking water ad libitum. The experiments wereapproved by the ethical committee of TransBIOTech (Levis, Qc, Canada).

Reagents

12-O tetradecanoyl phorbol 13-acetate (TPA, P-8139) and carrageenan(C-1138) were purchased from Sigma Chemical Co. (St-Louis, Mo., USA).

Preparation of the Thylakoid Extract

The thylakoid extract was obtained from spinach leaves (Spinaciaoleacea) as described in International patent publication WO 01/49305,the whole content of which is incorporated herein by reference. Thethylakoids integrity was evaluated by spectrophotometry (Beckman DU 640)(Lichtenthale 1987) and fluorimetry (Hansatech Instruments Ltd, England)(Maxwell 2000).

Protocol 1: TPA-induced Rat Ear Oedema

Male Wistar rats (180-200 g, Charles River) were fasted overnight (18h). Oedema was induced in the right ear of rats by topical applicationof 6 ug/ear of TPA in acetone (Yamamoto S et al. 1994). The left ear(control) received vehicle (acetone, 20 ul).

Six hours after TPA application, rats were anesthetized (pentobarbital;80 mg/kg) and a 6 mm diameter disc from each ear was removed with metalpunch. The swelling induced by TPA was assessed as the increase inthickness (in mm) of the right ear punch biopsy over that of the leftear and called the oedema index.

The thylakoid extract (25 mg/kg) was administered directly to theduodenum (5 ml/kg) via a catheter previously inserted into the duodenum.Physiology saline was administered for control groups (5 ml/kg).

Protocol 2: Carrageenan-induced Rat Paw Oedema

Male Wistar rats (180-200 g) which had been fasted overnight (18 h)received the thylakoid extract (25 mg/kg in sterile physiologic saline)by gavage (5 ml/kg) immediately prior to sub-plantar injection in theright hind paw of carrageenan (100 ul of 1% suspension in 0.9% saline)(Boughton-Smith et al. 1993),or by catheter for an in situ release as inprotocol 1.

Paw circumference was measured immediately prior to carrageenaninjection and also 5 h afterwards. Oedema was expressed as the increasedin paw circumference (in mm) measured after carrageenan injection andcompared to the pre-injection value for individual animals.

Statistical Analysis

Data are presented as mean ± standard error of the means. Meandifferences between groups were compared by t-test (SigmPlot 2001 forWindows Version 7.101).

Results

Effect of Thylakoids on TPA-induced Ear Oedema in Rats.

Topical application of TPA in control rats induced an increase in earthickness (50%) over 6 h (FIG. 1). Simultaneous administration ofthylakoids (25 mg/kg given directly into the duodenum via an insertedcatheter) reduced (45%) significantly ear oedema induced by TPA.

Effect of Thylakoids on Carrageenan-induced Rat Paw Oedema

The sub-plantar injection of carrageenan in control rats induced anincrease in paw circumference (5.63±1.29) over 5 h (FIG. 2).Simultaneous treatment with the thylakoid extract (25 mg/kg) directlyinto duodenum via a previously inserted catether or by gavage (5 ml/kg),inhibited oedema by 54% and 65%, respectively.

The above results show that the thylakoid extract can be administeredenterally or orally. In inflammation models like TPA-induced rat earoedema and carrageenan-induced rat paw oedema, a decrease of oedema ofabout 50% was observed at a dose of 25 mg/kg. Thus it is presumed that adose of 10 to 10000 mg p.o. per day of thylakoids could be used alone orin combination with any other adjuncted pharmaceutical compound. Theintended use is pharmaceutical as well as in food industry as foodsupplement, additive, preservative or as nutrient per se.

EXAMPLE 2 The Thylakoid Extract can be Formulated as a Product for OralUse

Materials and Methods

Materials

Three commercially available polymers were used for this study sodiumalginate, carboxymethyl cellulose low viscosity (CMC1) and carboxymethylcellulose high viscosity (CMC2). The complex PCT was given by PureCellTechnologies inc.

PCT Stability to Compression

First of all, PureCell Technologies inc. PTC was compressed as such,with any excipient, in order to evaluate the capacity of PCT to preserveits biological activity, following compression. Tablets of 200 mg madefrom PCT only were obtained by dry compression at 1, 2.5 and 5 T in aCarver hydraulic press using a punch of 9 mm diameter. The obtainedtablets were broken down to powder and sent to PureCell Technologiesinc. where the complex activity was tested.

PCT Stability to Compression in Presence of Polymeric Excipients

Tablets of 200 mg based on, one of the three polymers (alginate, CMCJ orCMC2) containing 20, 40 or 60% of PCT were obtained by dry compressionat 2.5 T in a Carver hydraulic press using a punch of 9 mm diameter. Theobtained tablets were sent to PureCell Technologies inc. where thecomplex activity was tested.

Tablet Behavior in Simulated Gastrointestinal Fluid

Two series of tablets of 200 mg were realized, one composed of one ofthe three polymers (alginate, CMC 1 or CMC2) without the PCT and theother based on one of the three polymers containing 20, 40 or 60% ofPCT. Tablets were obtained by dry compression at 2.5 T in a Carverhydraulic press with a 9 mm diameter punch.

The comportment of tablets was tested in simulated gastric fluid (SGF)and in simulated intestinal fluid (SIF). These medium were preparedaccording to U.S. Pharmacopeia (1990) with the difference that weomitted the addition of pepsin and pancreatin because none of thepolymers tested can be hydrolyzed by these enzymes. The medium wereprepared as follow:

-   -   For SGF an amount of 2 g sodium chloride and of 7 mL HC1 (37%)        were dissolved in sufficient water to make 1 L.    -   For SIF an amount of 6.8 g of monobasic potassium phosphate was        dissolved in 250 mL of water and a volume of 190 mL of 0.2 N        sodium hydroxide was added to the solution to adjust pH at 7.5.        Then, the solution was completed to 1 L to obtain the simulated        intestinal fluid solution.

Practically for gastro-intestinal comportment, tablets were placed in 50mL of SGF for 1 hour and then in 50 mL of SIF for 5 hours. The tablet'sbehavior was evaluated after each hour (glass adhesion, swelling,dissolution).

Results

PCT Stability to Compression

There is no effect of the compression force on the membrane integrity.The total carotene contents and the total chlorophyll contents were thesame and, as results, the ratio chlorophyll/carotene was unchanged (FIG.3).

The photosynthetic activity of the PCT was moderately affected by thecompression in fact about 35% of the activity was lost during thecompression with the mention that compression force do not seems toaffect the activity (FIG. 4).

PCT Stability to Compression in Presence of Polymer

The variation of the carotene and chlorophyll contents wasproportionally increased with the PCT contents of the tablet and in eachcase the ratio chlorophyll/carotene was unchanged. On the other side,there is a variation of the amount of pigment determined in tablethaving the same amount of PCT but formulated with different polymers(FIG. 5). In fact, for CMC1 a higher amount of pigments was detectedthan for CMC2, for which a higher amount of pigments than for alginatewas detected.

It appears that alginate led to lower amounts of pigments than CMCexcipients. As a possible explanation, the higher adhesive capacity ofalginate can retain part of the pigments, or disturb the assay. AmongCMC excipients, CMC1 (low viscosity) led to highest amounts of pigmentsdetected. Same effect of higher pigments retention on high viscosityCMC2 can explain this behaviour. However, differences among polymericexcipients are much lower in terms of total carotenoids, Chla/Chlb andChl/Car ratio.

Concerning the photosynthetic activity, for tablet containing 20% PCTCMC1 conserved more activity following by alginate and CMC2 indecreasing order. The activity increase was not strictly proportionalbut the growing was continuous with tablet contains. It looks like thePCT contents increased from 20% to 40 and 60% moderately increased thephotosynthetic activity (FIG. 6).

Tablet Behaviour in Simulated Gastrointestinal Fluid

The behaviour of tablets composed of polymers only is presented intable 1. During one hour incubation in SGF, alginate and CMC1 polymericmatrices have a slight swelling and stick to the glass, whereas CMC2,which sticks to the glass too, have a higher swelling volume. After onehour in SIF, all the polymeric tablets were surrounded by a gel andremained adhering to the glass. During the four next hours in SIF, thedifferent types of tablets were always adhered to the glass. Alginatecontinuing to swell, it start to dissolve after 4 hours in SIF and doesnot totally form a gel, even after 5 hours. CMC1 start to dissolve afteronly 2 hours in SIF; after 5 hour its dissolution was very advanced andit was completely under gel form. CMC2 have the highest swelling volumeand was completely under gel form but does not seem to dissolve. Otherauxiliary agents are to be added to improve CMC2 pellets dissolution.

The behaviour of tablets containing 20, 40 or 60% of PCT was similar tothose of the corresponding polymer without PCT. An additionalobservation was the liberation in SIF of the green PCT. With alginatethere is few PCT release which is not significantly increased at higherPCT loading. CMC2 forms a highly swollen gel, which releases few amountsof PCT and the release increased with the increase of PCT tabletloading. CMC1 dissolution helped the release of PCT, which ispractically totally released in 5 hours. Auxiliary agents may be addedto modulate the pellet dissolution, rate and time, and the thylakoidsrelease.

The invention being hereinabove described, it will be obvious that thesame be varied in many ways. Those skilled in the art recognize thatother and further changes and modifications may be made thereto withoutdeparting from the spirit of the invention, and it is intended that allsuch changes and modifications fall within the scope of the invention,as defined in the appended claims. TABLE 1 Tablets behavior during theincubation in simulating gastric fluid (SGF) and simulated intestinalfluid (SIF). 1 hour over SGF 1 hour SIF 2 hours SIF 3 hours SIF 4 hoursSIF 5 hours SIF night Alginate Glass yes yes yes yes yes yes Yesadhesion no no around around around Gel aspect around low low + +Swelling no no no no begin Tablets dissolution close to totallyTotally + + partially Totally CMC 2 Glass yes yes yes yes yes yes Yesadhesion no around around around Totally Gel aspect + ++ ++ ++ ++Swelling no no no no No Tablets dissolution close to totally totally ++++ no no CMC 1 Glass yes Yes yes yes yes yes Yes adhesion around Aroundaround Totally Gel aspect “hat” low + + Swelling no No begin totallyTablets dissolution close to totally close to totally totally + + +begin partially partially

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Bougthon-Smith N K, Deakin A M, Follenfant R L, Whittle B J, Garland LG. Role of oxygen radicals and arachidonic acid metabolites in thereverse passive Arthus reaction and carrageenin paw oedema in the rat.Br J Pharmacol 1993;110:896-902.

Purcell M. (1999), Procedure for preparing active plant extracts used totrap free radicals; the extracts and compounds and devices containingthem. Canadian patent CA 2293852.

Lichtenthaler H. K. (1987), Chlorophylls and carotenoids: Pigments ofPhotosynthetic Biomembranes In: Packer L. and Douce R. (eds.) Methods inEnzymology, vol 148 pp 350-382. Academic Press, London.

Maxwell Kate (2000), Chlorophyll fluorescence- a practical guide.Journal of experimental botany vol. 51 no 345. pp. 659-668.

US. Pharmacopeia National Formulary (1990), USP XXII, NF XVII, p. 1789,United States Pharmacopeial Convention Inc., Rockville (Md.)

1. A method for making an oral composition for treating or preventing adisease or disorder involving the formation of reactive oxygen speciesor inflammation, comprising combining purified thylakoids with acarrier, with the proviso that the carrier does not essentially consistsof water, physiological saline or propylene glycol.
 2. A method forpreventing oxidative damages to components of an oral composition,comprising adding purified thylakoids to said composition.
 3. The methodaccording to claim 1 or 2, wherein the oral composition is food or foodsupplement.
 4. A method for treating or preventing a disease or disorderinvolving the formation of reactive oxygen species or inflammation, in asubject, which comprises the step of orally administering an effectivedose of purified thylakoids.
 5. An oral composition comprising purifiedthylakoids and a carrier for oral ingestion or oral administration, withthe proviso that the carrier does not essentially consists of water,physiological saline or propylene glycol.
 6. The oral composition ofclaim 5, which is food or food supplement.
 7. The oral composition ofclaim 5, which is a medication selected from the group consisting of apellet, encapsulated granules and encapsulated powder.
 8. The oralcomposition of claim 5, wherein the carrier is present in an amount of0.01% to 95% (w/w).
 9. The method according to claim 1 or 2, wherein thepurified thylakoids are present in an amount which achieves a dosage of0.1 to 10 mg per Kg of a subject's body weight.
 10. The method asdefined in claim 4, wherein the purified thylakoids are present in anamount which achieves a dosage of 0.1 to 10 mg per Kg of a subject'sbody weight.
 11. The composition according to any one of claims 5 to 8,wherein the purified thylakoids are present in an amount which achievesa dosage of 0.1 to 10 mg per Kg of a subject's body weight.